Oct 16, 2023
Size Exclusion Chromatography with Multiangle Light Scattering (SEC-MALS)
- 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Protocol Citation: Minghao Chen, Xuefeng Ren 2023. Size Exclusion Chromatography with Multiangle Light Scattering (SEC-MALS). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkom4xv5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 16, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 89373
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350
Abstract
SEC-MALS
Concentrate the purified protein sample to 4-5 mg/ml
Instruments:
Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA)
Wyatt DAWN HELEOS-II MALS instrument
Wyatt Optilab rEX differential refractometer (Wyatt, Santa Barbara, CA)
WTC-050S5 size-exclusion column (Wyatt)
40 ul sample loop
Equilibrate the system and column with buffer containing:
25 mM HEPES pH 7.5, 200 mM NaCl, 1 mM MgCl2, 2 mM TCEP
Overnight
Turn on the laser 1 hour before the experiment.
Confirm the readout is stable
Run 5 mg/ml BSA to calibrate the system
Inject the sample and run (takes ~1 hour/sample)
Analyze the results by ASTRA V software (Wyatt).
MALS signals, combined with the protein concentration determined by refractive index, are used to calculate the molecular mass of the complex.